ol triricinoleate 35) and polysorbate 80 (Tween 80; polyoxyethylene-sorbitan-20-monooleate) are widely used as drug formulation vehicles, including for the taxane anticancer agents paclitaxel and docetaxel. A wealth of recent experimental data has indicated that both solubilisers are biologically and pharmacologically active compounds, and their use as drug formulation vehicles has been implicated in clinically important adverse effects, including acute hypersensitivity reactions and peripheral neuropathy. CrEL and Tween 80 have also been demonstrated to influence the disposition of solubilised drugs that are administered intravenously. The overall resulting effect is a highly increased systemic drug exposure and a simultaneously decreased clearance, leading to alteration in the pharmacodynamic characteristics of the solubilised drug. Kinetic experiments revealed that this effect is primarily caused by reduced cellular uptake of the drug from large spherical micellar-like structures with a highly hydrophobic interior, which act as the principal carrier of circulating drug. Within the central blood compartment, this results in a profound

Pharmacological effects of formulation vehicles : implications for cancer chemotherapy.

Publisher Summary This chapter focuses on the structure and function of alkyltransferase protein. O 6 -alkylguanine-DNA alkyltransferase is a remarkable protein that alone can, in a single step, remove adducts from DNA that are formed at the O 6 -position of guanine and the O 4 -position of thymine and can, thus, restore the original DNA. Production of such adducts is a major contributor to the toxic, mutagenic, and carcinogenic effects of alkylating agents. Alkyltransferase activity has been detected in many species, including microorganisms, insects, fish, and mammals. The unique activity of alkyltransferase suggests that it is an ideal target for biochemical modulation. The efficient repair of toxic lesions formed at the O 6 -position of guanine without additional enzymes or cofactors provides a less complex target to modulate than other DNA repair proteins. Furthermore, the high degree of correlation that exists between alkyltransferase activity and sensitivity to nitrosoureas indicates that elimination of this protein may reverse resistance in many cases. Two methods have been used to overcome alkylnitrosourea resistance by inactivation of alkyltransferase. One uses methylating agents that indirectly decrease alkyltransferase levels by introducing O 6 -methylguanine residues in DNA that are then repaired by the alkyltransferase. The second method uses direct alkyltransferase inactivators such as O 6 -methylguanine.

Structure, function, and inhibition of O6-alkylguanine-DNA alkyltransferase.

Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O(6)-methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O(6)-benzylguanine showed that, in gliomas, the apoptotic signal originates from O(6)-methylguanine (O(6)MeG) and that repair of O(6)MeG by MGMT prevents apoptosis. We further demonstrate that O(6)MeG-triggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O(6)MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O(6)MeG-induced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by gammaH2AX formation. Glioma cells mutated in DNA-PK(cs), which is involved in non-homologous end-joining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.

Apoptosis in malignant glioma cells triggered by the temozolomide-induced DNA lesion O6-methylguanine

BACKGROUND During the 1980s, the incidence of primary malignant brain and other central nervous system tumors (hereafter called brain cancer) was reported to be increasing among all age groups in the United States, while mortality was declining for persons younger than 65 years. We analyzed these data to provide updates on incidence and mortality trends for brain cancer in the United States and to examine these patterns in search of their causes. METHODS Data on incidence, overall and according to histology and anatomic site, and on relative survival were obtained from the Surveillance, Epidemiology, and End Results (SEER) Program of the National Cancer Institute for 1975 through 1995. Mortality data were obtained from the National Center for Health Statistics. Medicare procedure claims from the National Cancer Institute's SEER-Medicare database were used for imaging trends. Statistically significant changes in incidence trends were identified, and annual percent changes were computed for log linear models. RESULTS/CONCLUSIONS Rates stabilized for all age groups during the most recent period for which SEER data were available, except for the group containing individuals 85 years of age or older. Mortality trends continued to decline for the younger age groups, and the steep increases in mortality seen in the past for the elderly slowed substantially. Patterns differed by age group according to the site and grade of tumors between younger and older patients. During the last decade, use of computed tomography scans was relatively stable for those 65-74 years old but increased among those 85 years old or older. IMPLICATIONS Improvements in diagnosis and changes in the diagnosis and treatment of elderly patients provide likely explanations for the observed patterns in brain cancer trends.

Cancer surveillance series [corrected]: brain and other central nervous system cancers: recent trends in incidence and mortality.

A number of DNA-damaging chemotherapeutic agents attack the O6 position on guanine, forming the most potent cytotoxic DNA adducts known. The DNA repair enzyme O6-alkylguanine DNA alkyltransferase (AGT), encoded by the gene MGMT, repairs alkylation at this site and is responsible for protecting both tumor and normal cells from these agents. Cells and tissues vary greatly in AGT expression, not only between tissues but also between individuals. AGT activity correlates inversely with sensitivity to agents that form O6-alkylguanine DNA adducts, such as carmustine (BCNU), temozolomide, streptozotocin, and dacarbazine. The one exception is those tumors lacking mismatch repair, which renders them resistant to methylating agents. A recent study in patients with gliomas confirmed the correlation between low-level expression of the MGMT gene and response and survival after BCNU. An inhibitor to AGT, O6-benzylguanine (BG), depletes AGT in human tumors without associated toxicity and is now in phase II clin...

Clinical Relevance of MGMT in the Treatment of Cancer

Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransferases from the E. coli ada gene or from yeast were not affected. O6-Benzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltransferases at 200 microM in 30 min, slightly affected the Ada protein, and had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivation of the human alkyltransferase by O6-benzylguanine led to the formation of S-benzylcysteine in the protein and to the stoichiometric production of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6-benzylguanine inactivates the protein by acting as a substrate for alkyl transfer and by forming S-benzylcysteine at the acceptor site of the protein. The inability of O6-benzylguanine to inactivate the microbial alkyltransferases may be explained by steric constraints at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine

We have previously shown that O6-benzylguanine can be used to deplete cells of the DNA repair protein O6-alkylguanine-DNA alkyltransferase and to enhance the sensitivity of human glioma (SF767) and colon tumor (HT29) cells to the cytotoxic effects of alkylnitrosoureas. In the present study, the combination of O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was evaluated in vitro to determine the number of DNA interstrand cross-links formed and in vivo to compare the therapeutic index with that of BCNU alone. The number of DNA interstrand cross-links, as measured by alkaline elution, was increased in HT29 cells treated with 10 µmO6-benzylguanine for 2 h prior to BCNU exposure compared to cells treated with BCNU only. The number of single strand breaks was not increased by prior exposure to O6-benzylguanine. To evaluate the therapeutic index, HT29 and SF767 cells were grown as xenografts in nude mice and the tumor growth rate after treatment with BCNU alone was compared with the rate after treatment with O6-benzylguanine and BCNU. Treatment was administered i.p. when tumors reached 100–200 mm3. For animals bearing HT29 xenografts that were treated with 60 mg/kg O6-benzylguanine 1 h prior to 20 mg/kg BCNU, the average time for tumor volume to increase by 200% was 25 days, compared to 10 days for animals treated with 20 mg/kg BCNU alone. For animals bearing SF767 xenografts, the tumor growth of controls was not significantly different from that of animals treated with O6-benzylguanine alone or BCNU alone up to the maximally tolerated dose (50 mg/kg). For these 3 groups, the average time for tumors to reach 300 mm3 was 9–12 days. However, when animals were treated with 80 mg/kg O6-benzylguanine 1 h prior to receiving 20 mg/kg BCNU tumor size did not increase for at least 21 days. Our studies demonstrate that the therapeutic index of BCNU can be increased when given in combination with O6-benzylguanine.

https://cancerres.aacrjournals.org/content/canres/52/5/1171.full.pdf

Effect of O6-Benzylguanine on the Sensitivity of Human Tumor Xenografts to 1,3-Bis(2-chloroethyl)-1-nitrosourea and on DNA Interstrand Cross-Link Formation

A series of O6- and S6-substituted purine derivatives were tested for their ability to deplete the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) in cell-free extracts from HT29 colon tumor cells and intact HT29 cells The order of potency was O6-(p-Y-benzyl)-guanine (Y = H, F, Cl, and CH3) > O6-benzyl-2'-deoxyguanosine > O6-(p-Y-benzyl)guanosine (Y = H, Cl, and CH3) > or = a series of 9-substituted O6-benzylguanine derivatives > or = O6-allylguanine > O6-benzylhypoxanthine > O6-methylguanine A series of 7-substituted O6-benzylguanine derivatives, 2-amino-6-(p-Y-benzylthio)purine (Y = H, CH3), 2-amino-6-[(p-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, and 7-benzylguanine were inactive It is concluded that for efficient AGT depletion, an allyl or benzyl group attached through exocyclic oxygen at position 6 of a 2-aminopurine derivative is required Activity is preserved with a variety of substituent groups attached to position 9 while substitution at position 7 leads to a complete loss of activity

Structural features of substituted purine derivatives compatible with depletion of human O6-alkylguanine-DNA alkyltransferase

Experiments were carried out in mice and hamsters to determine whether the activity of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, in tissues and tumors was reduced by treatment with O6-benzylguanine in vivo. Following intraperitoneal injection of O6-benzylguanine, there was a rapid and complete loss of alkyltransferase activity in both livers and kidneys of mice and hamsters. The activity in mouse tissues was slowly restored, reaching pretreatment activities at 16 hr and 72 hr after injection of O6-benzylguanine at 10 mg/kg or 126 mg/kg, respectively. The activity in hamster liver was restored at a significantly lower rate, reaching less than 20% pretreatment activity 72 hr after treatment with 100 mg/kg of O6-benzylguanine. The efficient reduction of alkyltransferase activity by O6-benzylguanine was in sharp contrast to the inability of O6-methylguanine to bring about similar reductions. Activities dropped to about 55% of pretreatment activities in several mouse organs 4 hr after treatment with 126 mg/kg of O6-methylguanine compared to a more than 90% reduction in activity in animals after treatment with O6-benzylguanine. The sensitivity of SF767 cells to meCCNU after treatment with O6-benzylguanine was increased substantially. Furthermore, treatment of nude mice carrying SF767 tumor with 60 mg/kg of O6-benzylguanine prior to either 7.5 or 15 mg/kg of meCCNU led to significant inhibition of tumor growth. These studies indicate that O6-benzylguanine is a suitable compound for use in experiments to examine the role of the alkyltransferase protein in vivo in counteracting the effects of alkylating agents.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of mammalian O6-alkylguanine-DNA alkyltransferase in vivo by O6-benzylguanine and its effect on the sensitivity of a human glioma tumor to 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea.

Antisera raised in rabbits to three peptides corresponding to amino acid sequences found in human O6-alkylguanine-DNA alkyltransferase were used to study the fate of the alkyltransferase protein in human colon tumor cells after exposure to N-methyl-N'-nitro-N-nitrosoguanidine or to O6-benzylguanidine. Under these conditions, the alkyltransferase protein becomes inactivated, presumably by the conversion of its cysteine acceptor site to S-methylcysteine or S-benzylcysteine respectively. It was found that the protein was rapidly degraded after such inactivation both in intact cells and in cell-free extracts. It is probable that a conformational change in the protein is brought about by conversion of the alkyltransferase to the inactive form by alkylation of the cysteine acceptor site. This change may render the protein very sensitive to proteolytic degradation. The rapid degradation of the inactive form of the protein may serve as a signal for its resynthesis but in the short term ensures that its reactivation by regeneration of the cysteine acceptor site is unlikely to occur to any significant extent. The short half-life of the inactivated alkyltransferase protein makes it probable that measurement of the content of the alkyltransferase protein by immunohistochemistry, which is likely to measure the sum of the active and inactivated forms of the protein, will nevertheless yield an accurate estimation of the cellular capacity to repair O6-methylguanine provided that procedures with sufficient specificity and affinity can be developed.

Use of antibodies to human O6-alkylguanine-DNA alkyltransferase to study the content of this protein in cells treated with O6-benzylguanine or N-methyl-N'-nitro-N-nitrosoguanidine.

Dolan Me

Papers

Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine

Effect of O6-Benzylguanine on the Sensitivity of Human Tumor Xenografts to 1,3-Bis(2-chloroethyl)-1-nitrosourea and on DNA Interstrand Cross-Link Formation

Structural features of substituted purine derivatives compatible with depletion of human O6-alkylguanine-DNA alkyltransferase

Modulation of mammalian O6-alkylguanine-DNA alkyltransferase in vivo by O6-benzylguanine and its effect on the sensitivity of a human glioma tumor to 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea.

Use of antibodies to human O6-alkylguanine-DNA alkyltransferase to study the content of this protein in cells treated with O6-benzylguanine or N-methyl-N'-nitro-N-nitrosoguanidine.